Lactic acid bacteria isolated from Kazakh traditional fermented milk products affect the fermentation characteristics and sensory qualities of yogurt

Abstract Lactic acid bacteria (LAB) play a crucial role in the development of the taste, texture, and aroma of traditional fermented milk products. Five LABs from Kazakh traditionally prepared dairy products showed continuous subculture stability, as well as proper acidification and coagulation ability. They were identified as Pediococcus pentosaceus (1–5, 1–7), Enterococcus faecium (1–19), and Lactobacillus plantarum (1–12, 1–15). Their coagulation time and acidity values ranged from 5.97 to 12.78 h and 76.47 to 89.39°T. Yogurts prepared with L. plantarum were more condensed and textural integrity than those with P. pentosaceus and E. faecium. Determination of the volatile compound profiles suggested a higher diversity of volatile compounds than the control. The sensory evaluation presented positive overall sensory quality scores for the yogurts prepared with 1–12 and 1–15. The results provide additional information regarding the contributions of native LABs to the unique flavor and sensory qualities of traditionally prepared milk products. They may help to select starters or adjunct starters for developing distinctive, traditional nomadic fermented milk to satisfy consumer demand and increase market acceptability.

is dried by hanging it in a well-ventilated place). These products are rich in nutrients, typically recognizable by their unique taste, and are popular in local markets (Zuo et al., 2014).
Lactic acid bacteria (LAB) play a crucial role in the development of the taste, texture, and aroma of traditional fermented milk products.
They are widely distributed in nature and are well-known as starters for traditional fermented products (Bao et al., 2011). Multiple researchers have examined the LAB species composition of traditional fermented products and expanded their potential industrial application. Ren et al. (2017) demonstrated that Lactobacillus was the most commonly isolated LAB species from fermented yak milk in central Tibet. Enterococcus faecium was detected in all wooden vats used for the production of traditional stretched cheeses in Italy (Scatassa et al., 2015). Recently, several studies have also explored the contribution of relevant strains of native LAB to the sensory properties of traditional dairy products (Choobari et al., 2021;Guarrasi et al., 2017;Madhubasani et al., 2020;Medeiros et al., 2016;Tian, Shi, et al., 2019;Tian, Xu et al., 2019).
To the best of our knowledge, limited work has focused on the contribution of LAB to the organoleptic properties of traditional Kazakh dairy products. Therefore, LAB strains in Kazakh dairy products were isolated, identified, and their fermentation characteristics were evaluated with the aim of providing additional information to select starters or adjunct starters for developing distinctive traditional fermented milk products such as yogurt or cheese.

| Sampling
Five traditionally prepared samples (one cheese, one milk knot, two yogurts, and one vrum) were obtained from local Kazakh herding families living in Tianshan Mountain, Xinjiang, China. The samples were collected aseptically in sterile bags, kept in an ice-box container during transit to the laboratory, and stored at −20°C until analysis.

| LAB isolation
Samples were isolated using two selective media, MRS and M17 (Beijing Land Bridge Technology), under both aerobic and anaerobic conditions at 37 and 30°C for 72 h to obtain as many LAB strains as possible. Isolates were selected and purified according to previously published methods (Medeiros et al., 2016).
All isolates were verified as LAB using a combination of Gram reaction, catalase activity, and morphological analysis. Gram reactions were visualized using an optical microscope (Scope A1; Carl Zeiss Microscopy) under oil immersion at 100-fold magnification.
Colony morphology was recorded using a colony counter Scan1200 (Interscience International). Cocci, bacilli, or coccobacilli colonies that were gram-positive with a negative catalase result were included in the LAB group (Abosereh et al., 2016). Isolates were stored at −20°C in MRS broth supplemented with 20% (v/v) glycerol and only activated prior to testing by two sequential transfers in the same broth used in the experiments.

| Primary selection of LAB with milk fermentative characteristics
The microbial culture was inoculated at a level of 2 ml/100 ml in sterile reconstituted skim milk (120 g/L) (BD Biosciences Pharmingen) fortified with 6% sucrose and incubated at 42°C. Each strain was subcultured 10 times in sterile reconstituted skim milk. After 10 generations, the strains with continuous subculture stability, suitable acidification, and coagulation ability were selected for further analysis.
Amplification was performed in a Veriti 96-well thermal cycler (Life Technologies). The PCR temperature profile was as follows: 95°C pre-denaturation for 10 min, 35 cycles of 95°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 30 s, then a 72°C extension for 10 min. PCR products were sequenced by Invitrogen Shanghai Trading Co. Ltd.
The 16S rRNA gene nucleotide sequences of the five isolates were analyzed and identified using the BLAST program on the NCBI website (http//:www.ncbi.nlm.nih.gov/blast). Alignments were performed to construct a phylogenetic tree and compare similarities among the sequences using the neighbor-joining method in MEGA software version 6.0 (http://www.megas oftwa re.net) and bootstrapped with 1000 replicates (Tamura et al., 2013).

| Fermentation characterization of selected strains in yogurt manufacturing
Yogurt was made from whole milk (Sanyuan Dairy). The milk was homogenized, pasteurized at 95°C for 15 min, and cooled to 40°C before the selected microbial culture was inoculated (2 ml/100 ml).
Samples were incubated at 42°C until coagulation (until pH reached 4.6). The samples were immediately cooled in an ice-water bath and stored at 4°C for 12 h to mature and the acidity was recorded.
Texture, flavor compounds, and sensory qualities were analyzed.
Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480, (China General Microbiological Culture Collection Center, Beijing, China) preserved at the China-Australia Joint Research Center for Dairy Future Technology was used as the control.

| Acidification and coagulation analysis
The pH was measured using a PB-10 pH meter (Sartorius Scientific Instruments). Acidity was determined by titration with 0.1 N NaOH using phenolphthalein as an indicator, and the results were expressed in Thorner degrees (°T). Coagulation activity was defined as clotting after incubation at 42°C within 4-6 h (fast), 6-12 h (medium), or >12 h (slow) time frames (Ayad et al., 2004;Yi et al., 2011).

| Textural analysis
Yogurt texture was evaluated by the backward-extrusion test using a TA-XT Plus texture analyzer (Stable Micro Systems) equipped with a 5 kg loading cell (Mousavi et al., 2019;Wang et al., 2019). The parameters were modified from the Exponent template: 35 mm diameter cylinder probe, 1.0 mm/s test speed, 30 mm penetration distance, and 10 g surface trigger force. The tests were carried out on samples prepared in 125 ml containers (64 mm diameter and 70 mm height). Hardness (g), springness (%), consistency (g × s), and gumminess index (g) were calculated using the Exponent program, based on the instruction manual supplied by the manufacturer.

| Volatile compound analysis
Twenty grams of yogurt was weighed and mixed with 5 g NaCl in a 60 ml glass vial. Five grams of the mixture was placed in a 20 ml head-space vial sealed with a polytetrafluoroethylene-faced silicone septum (VWR). Extractions were carried out with a solid phase microextraction device (Supelco) containing a fused-silica fiber coated with a 50/30 μm layer of Divinylbenzene/Carboxen/ Polydimethylsiloxane. The vial was equilibrated at 60°C, centrifuged at 300 rpm for 2 min, and the fiber was exposed to the headspace over the sample for 30 min. The fiber was conditioned before adsorption by heating it in the gas chromatograph injection port at 250°C.
After adsorption, the fiber was removed from the vial and immediately inserted into the GC-MS injection port (Bezerra et al., 2017).
The temperature program was as follows: 3 min at 32°C, 12°C/min ramp to 48°C, hold for 10 min, 6°C/min ramp to 130°C, 10°C/min ramp to 200°C, 20°C/min ramp to 230°C, and hold for 2 min. The injection port temperature was 250°C, and the flow rate was 1.0 ml/ min. The mass spectrometer was operated in electron impact mode with a source temperature of 230°C, an ionization voltage of 70 eV, and a scan range from 29 to 400 m/z at 3.33 scans/s. Identification of the volatile compounds was based on comparison of their mass spectra with those from previously analyzed authentic compounds (spectra from the 14.0 NIST spectrum library). The retention index used a homologous series of alkanes (C6-C28; Sigma-Aldrich). To quantify the volatile compounds, 2-octanol was used as an internal standard. Results were represented as the percentage of each compound, thus allowing strain comparisons based on the relative contents of each compound, but not of their concentrations in sample cultures (Cheng et al., 2017;Guarrasi et al., 2017).

| Statistical analysis
All experiments were performed in triplicate and results were expressed as the mean ± SD. Statistical analyses were performed by IBM SPSS Statistics 20 (IBM Inc.). One-way analysis of variance was used to compare the means. Mean separations were performed by S-N-K (Newman-Keuls). Differences at p < .05 were considered significant. Additionally, principal component analysis (PCA) was performed with JMP Pro 16 software.

| Isolation and selection of the fermentative LAB
Nineteen LAB strains were selected from MRS and M17 plates according to morphological analysis of colony color, shape, and gloss.
Of these, 14 strains were identified as gram-positive and catalasenegative cocci; the other five were gram-positive and catalasenegative bacilli presumed to be LAB.

| Yogurt textural properties
Textural characteristics of the yogurts are listed in Table 1. Strain 1-12 showed the highest hardness (187.48 g), springness (69.10%), and consistency (3804.66 g × s) values, which were similar to those of the control, followed by those of strain 1-15, which also showed F I G U R E 1 (a) Strain evaluation by Gram staining of the five selected strains (×1000), and (b) phylogenetic tree analysis based on 16S rRNA gene sequences of the five selected isolates high hardness, springness, and consistency values. Strains 1-12, 1-15, and 1-7 showed similar gumminess values to that of the control (19.38 g). According to Cheng et al. (2017), texture is an essential aspect of yogurt quality and plays an important role in sensory evaluation and consumer acceptability. Yogurts fermented by strains 1-12 and 1-15 were condensed and had integrity, suggesting their potential in fermented dairy product preparation.

| Volatile compound profile
Determination of the volatile compound profiles suggested that the five strains produced a higher diversity of volatile compounds than the control (Table 2). Thirty-six volatile compounds produced by the five sample strains were detected and grouped into seven chemical categories: ketones, alcohols, aldehydes, acids, ethers, esters, and polyaromatic hydrocarbons.
The characteristic aroma of fermented milk products is extremely variable because it is strongly influenced by the enzymatic activities of indigenous and/or added microorganisms (Routray & Mishra, 2011;Tian, Shi, et al., 2019;Tian, Xu, et al., 2019). As shown in Table 2, ketones and acids were the most abundant compounds in the yogurt aromatic profiles, followed by alcohols and aldehydes.
Ketones are generated from the β-oxidation of acyl lipids and are a rich constituent of many dairy products (Matera et al., 2018;Tian, Shi, et al., 2019;Tian, Xu et al., 2019). These compounds have characteristic odors and low perception thresholds (Frank et al., 2004).
Acids can be generated through either lipolysis or glycolysis, but they are mainly generated through lactose metabolism (Hayaloglu et al., 2013). Acids are also used in the formation of other aromatic compounds such as ketones and alcohols (Delgado et al., 2010).
Acetic acid was the major acid detected in all five isolates (>13%).
Acetic acid produces a vinegar flavor and is associated with the slight tart taste of dairy products (Tian, Shi, et al., 2019;Tian, Xu, et al., 2019). However, the control generated a higher relative abundance (%) of hexanoic (34.40%) and octanoic (21.37%) acid than the isolates, with the exception of 1-12 (Lactobacillus plantarum); the relative abundances (%) of hexanoic and octanoic acid were 17.42% and 9.83%, respectively, which were slightly higher than those of the other four isolates. Butyric acid was also detected in all the yogurt samples. Hexanoic acid, which originates from lipolysis, produces a light cream flavor. Butyric and octanoic acids contribute to the characteristic flavor of cheeses (Delgado et al., 2010).
The classes of alcohols and aldehydes followed those of ketones and acids in terms of the relative abundance (%) of volatile compounds for the five isolates, and were not detected in the control strain yogurt. Furfuryl alcohol was the major alcohol present.
Although alcohols have a limited influence on flavor due to their high sensory thresholds, they represent an index of the fermentation process (Langler et al., 1967). Furfural was the only aldehyde detected in the yogurts.

TA B L E 2 (Continued)
represents the differences among the samples fermented by the six strains. The corresponding loading plot was illustrated in Figure 3b, which represent the relative importance of each volatile compound and the relationships between volatile compounds and samples (Zhang et al., 2020). PC1 and PC2 explained 30% and 24% of the total variation, respectively.
Samples prepared with the six strains could be clustered into three groups. The first group (1-12 and 1-15) was located in the negative region of PC1 and the positive region of PC2. 1-5, 1-7, and 1-19 were clustered into the second group. The third group only contained the control sample. This distribution of PCA also suggested the flavor uniqueness of the traditional Kazakh fermented dairy products.
As shown in Figure 3b, five flavor compounds (2-heptanone, furfural, furfural alcohol, and other two polyaromatic hydrocarbons) were located in the negative region of PC1 and the positive region of PC2. 2-Heptanone, furfural, and furfural alcohol are typical compounds detected in fermented milk products (Cheng, 2010). Although 1-5, 1-7, and 1-19 were clustered into same group, they were located in the positive region of PC1 and the negative region of PC2, as the control was. There were total of four flavor compounds in this region, including three acids and one ketone. Those compounds were also found in yogurt products (Cheng, 2010).

| Sensory qualities
Sensory evaluation results for the five yogurt samples are shown in Table 3. Yogurts made with strains 1-12 and 1-15 received positive overall sensory quality scores for a smooth yogurt gel appearance and relatively satisfactory fermented flavor. This evaluation may be a result of their good acidification and coagulation ability, condensed and textural integrity, and diverse and unique flavors.
These results suggest that strains 1-12 and 1-15 can be used as adjunct cultures with a control strain, or other commercial strains, for manufacturing fermented dairy products with a unique flavor (Crow et al., 2001;Leroy & De Vuyst, 2004).

| CON CLUS ION
Our results showed that the yogurts fermented by the five LAB that were isolated from fermented milk products in Xinjiang, China had the unique flavor and sensory qualities. Two isolates, strains 1-12 (Lactobacillus plantarum) and 1-15 (Lactobacillus plantarum), might be practically applied as adjunct cultures for the production of fermented milk products because of their desirable acidifying capacity, condensed texture, and pleasing flavor qualities. Creating starters by mixing these native strains with commercial LAB should be considered to develop distinctive, traditional nomadic fermented milk products to satisfy consumer demand and increase market acceptability. Finally, the authors were very grateful to all the panelists who participated in the study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.